Method and kit for separating double-stranded nucleic acid from a single-stranded/double-stranded mixture of nucleic acids

ABSTRACT

A method and a kit for separating double-stranded nucleic acid molecules from a mixture containing both single-stranded and double-stranded nucleic acid molecules. The method is particularly suitable for separating hybridized from unhybridized probe nucleic acid.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to a method and a kit for separatingdouble-stranded nucleic acid molecules from a mixture containing bothsingle-stranded and double-stranded nucleic acid molecules and isparticularly suitable for separating hybridized from unhybridized probenucleic acids.

2. Description of the Prior Art

An important tool in the rapidly-evolving fields of genetic engineering,environmental microbial contamination monitoring, and medicaldiagnostics is the use of DNA and RNA probes. DNA and RNA probes aresingle-stranded nucleic acid molecules generally synthesized byso-called gene machines or made using recombinant DNA methods Probes areconstructed so that the base (i.e., gene) sequences of the probe match(and lend themselves to hybridization with) complementary sequences on atarget molecule They are used for clinical diagnosis of geneticdisorders, cancer, and disease-causing bacteria, protozoans, andviruses. In the environmental field, they can be used to rapidlyidentify specific microbial contaminants. Research scientists use probesin recombinant DNA experiments and in gene activation studies.

Prior to use, each molecule of the probe is labelled with a marker, suchas a radioactive tag, in order to allow determination of when and wherehybridization has occurred.

Before the hybridized target nucleic acid molecules can be identifiedbased upon the presence of the labelled probe, the unhybridizedsingle-stranded labelled probe molecules must be separated out.Otherwise, this latter material will act as "background noise",inhibiting attempts to identify the hybridized material. Conventionallyutilized commercial methods for effecting this separation of hybridizedprobe molecules from unhybridized single-stranded probe moleculesusually involve immobilizing the target nucleic acid on a membrane whichis reactive with nucleic acids. Such membranes bind all nucleic acidsnonselectively. In view of this nonselectivity characteristic, theactive sites on the membrane must be blocked before probe nucleic acidis added. This blocking entails numerous steps and an incubation periodtypically of two hours. Thus, the membrane must be treated with variousblocking agents after the target DNA is affixed to prevent nonspecificadhesion of the probe nucleic acid so that washing the membrane afterhybridization will remove unhybridized probe. Alternatively, selectiveabsorption of double-stranded nucleic acids using hydroxylapatite isemployed. The hydroxylapatite serves to effectively separate double-fromsingle-stranded DNA. This capability results from hydroxylapatite'sselective affinity for double-stranded DNA under certain ionic strengthconditions.

Either radioactive or nonradioactive probes may be used with themembrane immobilization technique. In a nutshell, the radioactive probemethod first involves incubating a membrane, that has single-strandedtarget sequences attached, with a radioactively labelled probe (usuallyphosphorus-32 (³² P)) consisting of a single strand of DNA or RNA withbase sequences that are possibly complementary to the target sequencesbeing studied. The probe hybridizes with only those target nucleic acidscontaining a complementary nucleic acid sequence. After hybridization,the membrane is washed and hybrids are detected by autoradiography. Thepresence of characteristic hybrid nucleic acid on the autoradiogram isindicative of the presence of a specific target sequence. Typically, theassay of a hybridized radioactively labelled probe requires numeroussteps and 40 hours. The extensive time and effort result from thenecessity of binding target nucleic acid to the membrane, blocking theremaining sites on the membrane that would otherwise nonspecificallybind labelled probe, hybridizing for long periods of time because thehybridization reaction is a two-phase reaction, and washing numeroustimes to remove unhybridized probe. Developing the autoradiogramtypically takes 24 hours.

Hybridized probe may also be separated from unhybridized probe usinghydroxylapatite and appropriate ionic strength buffers (Kohne, D. E.,1984, Patent Cooperation Treaty WO 84/02721). This method, which hasonly been demonstrated with radioactive probes, is much faster. There isno binding of target DNA to an immobilization support, no blocking ofbinding sites on a support, fewer washes, and hybridization is morerapid since the reaction take place in a single phase. Commercial kitsusing hydroxylapatite require only 14 steps and 1.5 hours.

The radioactive method has not been well received in medical diagnosticslaboratories for several reasons. First, the high specific activityradioactive materials typically used in kits employing this method havea relatively short half life. Consequently, the complementary probe DNAmust be prepared just prior to the hybridization procedure. Secondly,the radioactive material creates more rigid handling problems andundesirable hazards. It is therefore advantageous in most cases toprovide a label which is less hazardous and prolongs the shelf life ofthe probe.

The development of non-radioactive labelling of nucleic acid probespresents an alternative. A typical non-radioactive system is based onthe incorporation of a biotinylated deoxyuridine triphosphate into theDNA probe by the nick translation procedure. The resultant biotinylatedDNA probe is stable and behaves as does a non-biotinylated DNA probe.The biotinylated DNA probe technique has been applied to the detectionof specific DNA and RNA sequences in fixed cells or in tissues followingin siru hybridizations. It has also been used to visualize probe nucleicacid hybridized to target nucleic acid immobilized on membranes. Thedetection of the hybridized biotinylated probe is accomplished by eitherfluorescent antibody or enzyme amplification techniques. A typicalnon-radioactive labelled probe assay such as an enzyme labelled probemethod using membrane-bound target DNA generally requires at least 32steps and 18 hours. More washes are required than in the case where aradioactive probe is used, but development of the signal is more rapid.Thus, although avoiding the problems of radioactivity, the conventionalenzyme-label method is cumbersome in view of the large number of stepsand the long test time generally attributable to the above-describedimmobilization of target nucleic acid on a membrane as the method toeffect separation of hybridized from unhybridized probe nucleic acid.

A number of suggestions have been made in the literature for alternativeseparation methods to recover the hybridized probe target molecules. Forexample, U.S. Pat. No. 4,599,303, which teaches a method of firsthybridizing and then forming covalent bonds between probe and target,discloses at column 2, lines 55-61 thereof, several procedures forseparating covalently crosslinked double-stranded probe-target complexfrom single-stranded probe. These procedures are described as includinggel filtration, hydroxylapatite chromatography, enzymatic digestion,alkaline hydrolysis, and photoreversal or chemical reversal ofuncrosslinked crosslinking molecules.

The gel filtration technique referred to in the '303 patent generallydenotes a column chromatography-type procedure whereby large moleculesin the assay mixture pass through channels in the gel, whereas thesmaller molecules elute at different velocities through the gel toeffect a separation. This gel filtration procedure is believed by thepresent inventors to be slow, cumbersome, and subject to failure since,with sufficient elution, the entire assay mixture will pass through thecolumn.

In view of the above, new methods for separating single-stranded probenucleic acid from double-stranded probe/target nucleic acid moleculesare expected to be highly desired in a commercial setting, particularlyin the medical diagnostics and environmental monitoring fields. Morespecifically, the development of a simpler such technique employablewith either radioactive or non-radioactive labelled probes for clinicaldiagnosis and environmental monitoring would undoubtedly enhance thistechnique and will have a competitive advantage relative to currentapproaches.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a method for separatingdouble-stranded nucleic acid molecules from single-stranded nucleic acidmolecules which comprises ultrafiltration of a liquid mixture containingsaid double-stranded nucleic acid molecules and said single-strandednucleic acid molecules through an ultrafiltration membrane having a poresize within a range sufficient to pass said single-stranded nucleic acidmolecules but to retain said double-stranded molecules on said filter.As used herein, the terms "ultrafiltration" and "ultrafiltering" denotefiltration using a membrane filter having a sufficiently small pore sizesuch that macromolecules of a particular molecular weight, typicallyfrom 50,000 to 1,000,000 daltons, are retained by the filter.

In another aspect, the present invention relates to a method fordetermining the presence of specific nucleic acid base sequences insingle-stranded nucleic acid target molecules comprising the steps of:

(a) providing single-stranded labelled nucleic acid probe moleculeshaving an essentially complementary base sequence to a defined region inthe single-stranded target molecules;

(b) hybridizing at least a portion of said single-stranded labellednucleic acid probe molecules to said defined region in at least aportion of said target molecules, thereby forming a mixture ofhybridized molecules and single-stranded molecules;

(c) ultrafiltering said mixture using a membrane filter having amolecular weight cutoff sufficient to pass said portion of saidsingle-stranded labelled nucleic acid probe molecules through saidfilter but retain said hybridized molecules on said filter; and

(d) measuring the amount of said portion of said single-strandedlabelled nucleic acid probe molecules in said hybridized molecules orthe amount of single-stranded labelled probe molecules that pass throughsaid membrane filter and did not hybridize with target nucleic acidmolecules.

In still another aspect, the present invention relates to a kit foreffecting separation of labelled probe nucleic acid molecules fromhybridized probe/target nucleic acid molecules comprising:

(a) a sample of labelled probe nucleic acid molecules and

(b) a membrane filter having a pore size within a range such thatunhybridized labelled probe nucleic acid molecules pass through saidmembrane filter whereas labelled probe nucleic acid molecules hybridizedwith compatible target nucleic acid molecules are retained on saidmembrane filter.

These and other aspects of the invention will be readily apparent uponreading the detailed description which follows.

DETAILED DESCRIPTION OF THE INVENTION

The target DNA or RNA whose specific base, a portion of whose sequenceis generally known, is referred to herein as a target. Thepolynucleotide containing the label and expected to have a base sequencecomplementary to the target is referred to herein as a probe. Thejoining together of both target and complementary probe polynucleotideby the mechanism of base pairing through hydrogen bonds between purineand pyrimidine bases is referred to herein as hybridization and theresultant complex is termed a hybridized nucleic acid molecule orhybridized probe/target molecule.

The nucleic acid probe will consist of chemically synthesized orbiologically prepared DNA or RNA polynucleotides from 10 to 2000 basesin length or longer single-stranded sequences including messenger RNAsand single-stranded DNA or RNA. If synthesized, the single-stranded DNAor RNA probe is fabricated so that its nucleic acid base sequence iscomplementary to a region of the bacterial, viral, or mammalianchromosome target sequence.

Alternatively, probe DNA or RNA can be isolated from biological sourcesand subsequently reacted with a labelling reagent of interest.Single-stranded DNA can be obtained directly from single-stranded viralgenomes such as M13 or ΦX174 or indirectly from double-stranded genomesor plasmids by strand separation. The size of such a probe will becontrolled by enzymatic processing including exonuclease treatment ofsingle-stranded DNA and restriction or Bal 31 digestion ofdouble-stranded DNA. In another alternative, the DNA probe can also beprepared enzymatically from appropriate nucleic acid substrates. Forexample, DNA could be obtained from mRNA using reverse transcriptase.RNA probes can be directly obtained from biological sources in the formof viral genomes (R17, F2, QB) or mRNA. Alternatively, the RNA can beenzymatically synthesized in vitro from appropriate templates. Forexample, phage RNA polymerase catalyzed transcription of adouble-stranded DNA template such as a sequence cloned next to a phagepromoter in an appropriate cloning vector would generate probe RNA.

The probe will normally have at least 25 bases, more usually at leastseveral hundred bases, and may have up to about 10,000 bases or more,usually having not more than about 5,000 bases. The probe sequence willbe at least substantially complementary to a sequence characteristic ofthe organism or gene of interest. The probe need not have perfectcomplementarity to the sequence to which it hybridizes; there may be 30percent or more of mismatched pairs.

The probe may be obtained from messenger RNA, from ribosomal RNA, orfrom cDNA obtained by reverse transcription of messenger RNA orribosomal RNA with reverse trancriptase. Probe may also be obtained bycleavage of the genome, conveniently by endonuclease digestion, followedby cloning of the gene or gene fragment in accordance with knowntechniques. See, for example, Kornberg, DNA Replication, W. H. Freemenand Co., San Francisco, 1980, pp 670-679; So et al, Infect. Immun.21:405-411, 1978. After isolation and characterization of the desiredgene or DNA fragment, the gene or DNA fragment may be used forpreparation of the probe or transcribed for production of RNA, which maythen be used for preparation of the probe.

For the most part, the polynucleotide probe will be labelled with anatom or inorganic radical, most commonly using radionuclides, but alsoperhaps heavy metals. However, in some situations it may be feasible toemploy an antibody which will bind specifically to the probe hybridizedto the single-stranded target DNA. In this instance, the antibody wouldbe labelled to allow for detection. The same types of labels which areused for the probe may also be bound to the antibody in accordance withknown techniques.

If desired, a radioactive label may be employed. Radioactive labelsinclude ³² P, ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C, or the like. Any radioactive labelmay be employed which provides for an adequate signal and has sufficienthalf life. Other labels include ligands, which can serve as a specificbinding member to a labelled antibody or to a specific binding protein,fluorescers, chemiluminescers, enzymes, antibodies which can serve as aspecific binding site for a labelled ligand, and the like. A widevariety of labels have been employed in immunoassays which can readilybe employed in the present assay. The choice of the label will begoverned by the effect of the label on the rate of hybridization andbinding of the probe to the target DNA. It will be necessary that thelabel provide sufficient sensitivity to detect the amount of DNAavailable for hybridization. Other considerations will be ease ofsynthesis of the probe, readily available instrumentation, ability toautomate, convenience, and the like.

The manner in which the label is bound to the probe will vary dependingupon the nature of the label. For a radioactive label, a wide variety oftechniques can be employed. Commonly employed is nick translation withan -³² P-dNTP or terminal phosphate hydrolysis with alkaline phosphatasefollowed by labelling with radioactive ³² P employing -³² P-NTP and T4polynucleotide kinase. Alternatively, nucleotids can be synthesizedwhere one or more of the elements present are replaced with aradioactive isotope, e.g., hydrogen with tritium. If desired, extensionsof the probe strand with a simple repetition of one nucleotide (andtherefore unlikely to be complementary to any target sequence) can beused to enhance the concentration of hybridized label.

Where other radioactive compounds are involved, various linking groupscan be employed. A terminal hydroxyl can be esterified with inorganicacids, e.g., ³² P phosphate, or ¹⁴ C organic acids, or else esterifiedto provide linking groups to the label. Alternatively, intermediatebases may be substituted with activatable linking groups which can thenbe linked to a label.

Ligands and antiligands may be varied widely. Where a ligand has anatural receptor, namely ligands such as biotin, thyroxine, andcortisol, these ligands can be used in conjunction with labellednaturally occurring receptors. Alternatively, any compound can be used,either haptenic or antigenic, in combination with an antibody.

Enzymes of interest as labels will primarily be hydrolases, particularlyesterases and glycosidases, or oxidoreductases, particularlyperoxidases. Fluorescent compounds include fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.Chemiluminescers include luciferin, and 2,3-dihydrophthalazinedioners,e.g., luminol.

The amount of labelled probe which is present in the hybridizationsolution will vary widely, depending upon the nature of the label, andthe stringency of the hybridization. Generally, substantial excessesover a stoichiometric amount of the probe will be employed to enhancethe rate of hybridization and to allow the quantifying of the amount oftarget sequences present.

In the hybridization, the nucleic acid, for example, from a blood,tissue, cell sample, or microorganism from an environmental sample isreacted with the probe under conditions where hybridization of the probewith the target will occur. The probe contains a label molecule whichcan be a radioactive nuclide, chromogenic, fluorogenic, luminescent dyemolecule, magnetic particle, or an enzyme system capable of generating achromogenic, fluorogenic, and/or luminescent product via appropriatesubstrates.

The particular hybridization technique employed is not a criticalelement of the present invention. Various hybridization solutions may beemployed, comprising from about 20 to 60, preferably 40 to 50, volumepercent of an inert polar organic solvent. A common hybridizationsolution employs about 50 percent formamide, about 0.05 to 0.5 M sodiumphosphate, and minor amounts of EDTA. Other additives may also beincluded, such as dextran sulfate of from about 100 to 1,000 dal and inan amount of from about 8 to 15 weight percent of the hybridizationsolution. Alternatively, aqueous solutions containing these salts andpolymers and free of organic solvents such as formamide may be employed.The hybridization time employed can be one-half hour or less up toseveral hours or more as desired.

The more stringent these conditions, the greater the complementaritythat is required for hybridization between the probe and the targetnucleic acid.

The extent of hybridization is affected by various factors, includingtemperature, probe concentration, probe length, ionic strength, time,and the like. As an illustrative example, the extent of hybridizationcan be varied by changing the polarity of the reactant solution bymanipulating the concentration of formamide in the range of 0 to 50percent. Alternatively, temperatures can be varied in the range of about20° C. to 85° C., usually 30° C. to 75° C. Generally, atmosphericpressure is employed.

Detection of the hybridized probe will normally be accomplished bymeasuring the amount of the label on the double-stranded molecule afterhybridization. Various methods or protocols may be employed in measuringthe amount of the labels on the hybridized probe/target molecules. Theseprotocols include, for example, autoradiography, detection ofradioactive decay in a scintillation counter or using a geiger counter,chemiluminescent assays, bioluminescent assays, and assays of enzymeslinked either directly or indirectly to the probe, among others.

The hybridized labelled probe/target is separated from unhybridizedlabelled probe by ultrafiltration. This ultrafiltration is preferablyconducted while centrifuging the mixture containing hybridizedprobe/target, thereby expediting the ultrafiltration process.Ultrafiltration is suitably effected using a membrane filter constructedof any solid network material such as, for example, cellulosic, acrylic,or other material that will not bind either nucleic acid or proteinnonspecifically. Centrifugation, if used as a driving force for theultrafiltration, is generally applied at 1000 to 3000 rpms at between500 and 2000 RCF (relative centrifugal force), e.g. in a fixed-anglecentrifuge rotor.

Alternatively, more conventional ultrafiltration using pressure-drivenmembrane separation can be employed with pressures between 5 and 20atmospheres.

The discovery by virtue of the present invention that ultrafiltration ona membrane filter can be used to separate single-stranded (e.g.,unhybridized) from double-stranded (e.g., hybridized) nucleic acids isparticularly surprising in view of the similar structural configurationof these moieties. Without wishing to be bound by any particular theory,both single-stranded and double-stranded nucleic acids are commonlybelieved to be linear polymers or to have, at most, some secondarystructure, albeir often transitory. They are not globular inconfiguration like undenaturated proteins. In fact, it is more likelythat single-stranded nucleic acid, particularly RNA, has more secondarystructure than double-stranded material. This is due to internalhomologies in single-stranded material that causes intra-strandedbase-pairing with resultant double-stranded regions and interspersedloops of unpaired sections. It was of considerable surprise, therefore,to discover that ultrafiltration could be used to separate bound fromunbound probes since ultrafilters, although fabricated and calibrated onthe basis of the molecular weight of globular proteins retained,actually separate on the basis of the configuration of the molecules.Our discovery is as unexpected as finding that a spaghetti colanderwould pass certain forms (and retain slightly wider forms) of pasta,even though the colander's holes in both cases are much bigger than thediameter of both forms of pasta.

Ultrafiltration membranes having a molecular weight cutoff (MWCO) withina range of between about 50,000 and about 1,000,000 daltons aregenerally useful within the scope of the present invention. An optimalcutoff range is dependent upon the size of the specific probe and targetnucleic acids selected. For example, small probes with radioactivesignals are expected to have a preferred MWCO of about 100,000 or lower,whereas the preferred MWCO for probes with an enzyme label is betweenabout 100,000 and about 500,000, more preferably between about 200,000and about 300,000 daltons.

Preferred materials for membrane construction for filters useful in thepresent invention are those which are hydrophilic and have lownonspecific binding of proteins or nucleic acids. Membranes fabricatedof acrylic, cellulosic, or regenerated cellulose compositions are mostpreferred. Membranes fabricated of aromatic amides or polysulfones arealso expected to be useful within the scope of the present invention.Suitable commercially available ultrafiltration membranes can beobtained from Amicon Corporation, Micro Filtration Systems, Schleicherand Schuell, Ultra-Pore Inc., and Millipore Corporation.

Normally, the hybridization and subsequent ultrafiltration steps forisolation of the hybridized labelled probe/target molecules are done insolution without the need for electrophoretic gel separations orblotting procedures. The simplicity of the required manipulations, highsensitivity, and low background of the procedure have clear advantage toother hybridization and isolation assays.

The present invention provides a simple diagnostic method for detectionof the pathogenic origin of disease, for detection of genetic anomalies,and for monitoring for microbial contamination. The method findsparticular application in infectious and/or genetic disease diagnosis,epidemiology, and water and food monitoring. The method is reasonablyrapid, taking about three hours, has a simple protocol, as few assixteen steps, has reagents which can be standardized and provided incommercial kits, and allows for rapid screening of a large number ofsamples. In practice, samples can be taken through part of the protocolin "the field" and conveniently returned to a distant laboratory forfinal completion of the diagnosis.

In carrying out the method of the present invention, a sample suspectedof containing the microbe may be used directly or cultivated underconditions where organism growth provides high multiplication of theorganism's nucleic acids. A tissue sample suspected of having anomalousgene activity would be used directly. After treating the target toprovide single-stranded genomic nucleic acid, the single-stranded DNA orRNA is hybridized with a labelled probe polynucleoride having acomplementary base sequence. This hybridization generally takes place inthe presence of an excess of probe relative to the amount of theknown-sequence target to be hybridized. For example, a 100 fold to 1,000fold excess of probe to specific target sequences will allow rapidhybridization of all target sequences. Such an excess also allowsquantitative analysis of numbers of contaminating organisms in singletest.

As stated above, the minimum number of components of a kit in accordancewith the present invention is a sample of labelled probe and a membranefilter. However, it is preferred that the kit also contain a suitablelysing system for lysing of cells or viruses to provide target nucleicacid molecules for hybridization with the labelled probe nucleic acidmolecules. The kit also preferably contains a wash solution (e.g., aphosphate-buffered 50 percent formamide solution) for washingunhybridized labelled probe nucleic acid through the membrane filter. Ifthe probe is enzyme labelled, the kit also preferably contains an enzymesubstrate and buffer solution to optimize the enzyme's catalyticactivity and to allow signal development and/or enhancement of the labelfor identification of the hybridized probe/target nucleic acidmolecules.

Microorganisms which may be detected and identified include bacteria,viruses, fungi, protozoa, algae, etc. Among microorganisms are bacteria,such as gram negative bacilli, e.g. Escherichia, Vibrio, Yersinia,Klebsiella, and Salmonella. Species include E. coli, Vibrio cholerae,Haemophilus ducrei, Legionella pneumophila. Other microorganisms ofinterest are those difficult to cultivate such as the HIV virus, genitalHerpes virus, Norwalk Agent virus, Rotavirus, and Giardia.

The following examples are intended to illustrate, but in no way limit,the scope of the present invention.

EXAMPLE 1 HYBRIDIZATION OF PROBE DNA WITH VARIOUS BACTERIAL NUCLEICACIDS AND ULTRAFILTRATION TO ISOLATE THE HYBRIDIZED MATERIAL

In order to test the efficacy of the hybridization and ultrafiltrationmethod of the present invention, several tests were conducted. The testswere designed to show that retention of the probe nucleic acid by theultrafiltration membrane depended on hybridization with target nucleicacid. The tests were done with three different types of probes: asingle-stranded phage genome (used in the present example), an RNAtranscript of a cloned sequence specific for Bactericides target DNA(see EXAMPLE 2), and a copy DNA (cDNA) probe made by reversetranscription of bacterial ribosomal RNA (rRNA) (see EXAMPLE 3). Thesignal system most commonly used in these experiments is an enzymecatalyzed colorimetric reaction with the enzyme, horseradish peroxidase,covalently attached to the probe. However, also included are experimentsin which the signal was an enzyme catalyzed colorimetric reaction inwhich the probe and the enzyme are both biotinylated and the enzyme isindirectly attached via a biotin-avidinbiotin linkage as well asexperiments in which the probe was radioactive. In the first example theprobe was a phage genome and the signal was an enzyme linked eitherdirectly or indirectly to the probe. Tests were conducted in accordancewith the following sequence of steps:

A) Chromosomal DNA was isolated and purified from bacterial cells fromBacillus subtilis or Escherichia coli (both a laboratory strain and afecal isolate) using standard procedures (Maniatis et al, MolecularCloning: A laboratory Manual, Cold Spring Harbor Laboratory, New York,1982). DNA purification was taken through phenol/chloroform extraction,ether extraction, and ethanol precipitation. The DNA was put intoaqueous solution and analyzed quantitatively using spectrophotometry sothat in all experiments the same quantity of DNA was used in control andexperimental hybridizations.

B) A DNA probe was made that consisted of the entire M13mp18 bacterialvirus (phage) genome. This single-stranded phage was chosen as a modelprobe because the genome is easy to isolate, and there are well workedout methods for cloning sequences into this phage (Heidecker et al., AVersatile Primer for DNA Sequencing in the M13mp2 Cloning System, Gene10: 59-73, 1980; Hackett et al, An Introduction to Recombinant DNATechniques, The Benjamin/Cummings Publishing Company, Inc., Menlo Park,California, 1984). Methods for easily using the inserted portion of suchrecombinant phage as a probe have been described (Brown et al.,Sensitive Detection of RNA Using Strand-Specific M13 Probes, Gene 20:139-144, 1982). Phage particles were grown and isolated and the DNApurified by phenol extraction as in DNA sequencing experiments (Hackettet al., An Introduction to Recombinant DNA Techniques, TheBenjamin/Cummings Publishing Company, Inc., Menlo Park, California,1984).

The probe was labelled by directly attaching the enzyme horseradishperoxidase (I.U.B.1.11.1.7) covalently using the protocol of Renz andKurz, A Colorimetric Method for DNA Hybridization, Nucleic AcidsResearch 12: 3435-3444, 1984. This probe-enzyme complex was then used ina hybridization reaction with DNA from the bacterial nucleic acids asdescribed below. The final concentration of the probe nucleic acid was1.3 μg/ml in the final 380 μl hybridization solution. Hybridization wascarried out in a microcentrifuge tube.

C) Chromosomal DNA was placed in an aqueous 0.75 M phosphate buffersolution with a pH of 6.8, that was 50 percent formamide. The finalconcentration of the chromosomal DNA was 118 μg/ml in a total volume of380 μl. The DNA was then melted into single strands by heating to 85° C.for four minutes. The DNA solution was cooled to 40° C. and labelledprobe was added and hybridized at this temperature for three hours.

D) After completion of hybridization, the entire 380 μl hybridizationmixture was loaded into an MPS-1 micropartition system, an apparatus formembrane ultrafiltration supplied by Amicon that uses a slow speed(clinical type) centrifuge. The MPS-1 contained an Amicon XM300ultrafiltration membrane made of acrylic material which, bymanufacturer's specifications, has a molecular weight cutoff of 300,000daltons. The apparatus was centrifuged at low speed in a DamonWhisperfuge Model 1385 in a fixed angle rotor to near dryness. Thisspeed is 2000 rpm and results in 500 RCF. The microcentrifuge tube wasthen washed with a 50 percent formamide, 0.75 M phosphate buffersolution, with a pH of 6.8 that was prewarmed to 40° C. The wash wasloaded into the same ultrafiltration membrane apparatus and again spunto near dryness. The filter was then washed twice more with the samesolution, and then twice with a solution of 50 mM citrate buffer pH 4,warmed to 37° C. The second citrate buffer wash was allowed to stand onthe membrane for 20 minutes at 37° C. prior to centrifugation in orderto fully reactivate the peroxidase enzyme. One milliliter of a solutioncontaining 67.5 μg/ml of 2,2'-azino-di-3-ethylbenzthiazoline sulfonicacid and .006 percent H₂ O₂ in 50 mM citrate buffer pH 4 was added tothe apparatus. If peroxidase is present, it converts the substrate intoa soluble blue dye. This solution was pipetted from the ultrafiltrationmembrane apparatus into 2 ml of citrate buffer in a spectrophotometrictube with a 1 cm path length and the optical density at 405_(nm)determined at various times.

                  TABLE I                                                         ______________________________________                                                         OD* 405.sub.nm after 45 Min.                                 Bacterial Source Peroxidase Reaction                                          of Target DNA    (Replicate Samples)                                          ______________________________________                                        No target DNA added                                                                            0.11, 0.11                                                   B. subtilis      0.10, 0.20                                                   E. coli - fecal isolate                                                                        0.55, 0.58                                                   E. coli - laboratory strain                                                                    0.05, 0.05                                                   ______________________________________                                         *Optical density at an incident light of 405.sub.nm wavelength using a        Spec 20 spectrophotometer with a 1 cm path length is a measurement of         color change due to enzyme activity.                                     

The optical density values given in TABLE I above provide quantitativeinstrumental measurement of the extent of color change by virtue ofenzymatic activity. Color is seen (as indicated by optical density at⁴⁰⁵ nm of greater than a 0.1 to 0.2 background value) only if enzyme ispresent to catalyze a colorimetric reaction. The enzyme is covalentlyattached to the probe, so color is an indication that the probe isretained by the membrane. The probe is specific for E. coli fecalisolate DNA. The B. subtilis and E. coli laboratory strain DNA serve asnegative controls in this experiment.

The data presented in TABLE I demonstrates that the retention of probeafter hybridization by the ultrafiltration membrane depends on thepresence of a specific target DNA (in this case, the fecal isolate formof E. coli). This specificity indicates that hybridization isresponsible for probe retention by the filter.

In numerous analogous experiments, the control tube in which no targetDNA was added to the hybridization reaction consistently yielded resultssimilar to that provided by the tube containing the heterologous (B.subtilis) non-target DNA. Therefore, in some subsequent experiments theB. subtilis target nucleic acid tube served as a comparative example.

In a subsequent experiment, this probe showed the same specificity whencrude lysates rather than purified bacterial DNA was used. To make crudelysates, the lysozyme, boiling water method of Maniatis et al, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York,1980, was used. The debris from the lysis was pelleted and thesupernatant used as a source of target nucleic acid. We also found thathybridization for 30 minutes was sufficient.

When the enzyme labelled M13mp18 probe was replaced with a biotinylatedM13mp18 probe, it shows the same specificity as a probe that has theenzyme directly attached. The biotinylated enzyme was added after bothhybridization and a stringent wash was completed. The fact thathybridized probe was retained even though it had no enzyme attachedprior to washing indicates that retention by the ultrafiltrationmembrane does not appear to be due to some non-specific interactionbetween material in the target DNA preparation and the signal systemenzyme.

EXAMPLE 2 HYBRIDIZATION OF PROBE RNA WITH VARIOUS BACTERIAL NUCLEICACIDS AND ULTRAFILTRATION TO ISOLATE THE HYBRIDIZED MATERIAL

This example was conducted to test the extent of probe retention by thefilter membrane after hybridization using an RNA probe. Several testswere conducted in accordance with the following procedural steps:

A) Chromosomal DNA was isolated and purified from bacterial cells fromBacillus subtilis, Bacteroides fragilis, Bacteroides thetaiotaomicron,or Escherichia coli using standard procedures as in EXAMPLE 1 above.

B) An RNA probe was made by transcribing a Bacteroides-specific sequencethat had been cloned into the cloning vector pGEMI right next to the T7promoter. The vector was cut with the restriction endonuclease Hind IIIand the transcript made using T7 RNA polymerase. The transcript waslabelled with the enzyme horseradish peroxidase (I.U.B.1.11.1.7) as inEXAMPLE 1. This probe-enzyme complex was then used in a hybridizationreaction with DNA from the bacterial nucleic acids as described below.The final concentration of the probe nucleic acid was 2.6 μg/ml in thefinal 100 μl hybridization solution. Hybridization was carried out in amicrocentrifuge tube.

C) Chromosomal DNA was placed in an aqueous 0.12 M phosphate buffersolution with a pH of 6.8, that was 50 percent formamide. The finalconcentration of the chromosomal DNA was 200 μg/ml in a total volume of100 μl. The DNA was then melted into single strands by heating to 85° C.for four minutes. The DNA solution was cooled to 47° C. and labelledprobe was added and hybridized at this temperature for three hours.

D) After completion of hybridization, the entire 100 μl hybridizationmixture was loaded into an MPS-1 apparatus containing an Amicon XM300ultrafiltration membrane as in EXAMPLE 1. The apparatus was centrifugedat low speed as in EXAMPLE 1. The microcentrifuge tube was then washedas before but with 0.12 M phosphate buffer solution prewarmed to 47° C.and then washed twice with a solution of 50 mM citrate buffer pH 4, asbefore. The dye was the same as used in EXAMPLE 1 and dye developmentwas read in a Spec 20 as in EXAMPLE 1.

Following the above procedure, several replicate experiments usingdifferent probe and target DNA reagent preparations providedhybridization results as shown by the representative data give in TABLEII below.

                  TABLE II                                                        ______________________________________                                                        OD* 405 after 21/2 hour                                       Bacterial Source                                                                              peroxidase reaction                                           of Target DNA   (replicate samples)                                           ______________________________________                                        B. subtilis     0.12, 0.10                                                    E. coli         0.10, 0.10                                                    B. thetaiotaomicron                                                                           0.35, 0.24                                                    B. fragilis     0.58, 0.44                                                    ______________________________________                                         *Optical density at an incident light of 405 .sub.nm wavelength using a       Spec 20 spectrophotometer with 1 cm path length.                         

The optical density values given in TABLE II above provide quantitativeinstrumental measurement of the extent of color change by virtue ofenzymatic activity. Color is seen (as indicated by optical density at⁴⁰⁵ nm of greater than a 0.1 to 0.2 background value) only if enzyme ispresent to catalyze a colorimetric reaction. The enzyme is covalentlyattached to the probe, so color is an indication that the probe isretained by the membrane. The probe is specific for Bacteroides DNA. TheB. subtilis and E. coli DNA serve as negative controls in thisexperiment. These data therefore show that the specificity of thehybridization reaction between the enzyme-labelled probe and the targetDNA results in specific retention of probe by the filter.

In subsequent experiments, crude lysates were made of bacterial culturesusing a lysozyme, boiling water method as in EXAMPLE 1 and shorter 30minute hybridization incubation periods were used. Hybridization wasstill specific as shown in TABLE III below.

                  TABLE III                                                       ______________________________________                                        STRAIN                                                                        B.              B.          E.                                                fragilis        thetaiotaomicron                                                                          coli                                              ______________________________________                                        OD.sub.405                                                                            1.1         0.91        0.26                                          ______________________________________                                    

Readings are ^(OD) 405, indicating extent of enzyme catalyzedcolorimetric reaction. Reaction time: 30 minutes.

These data show that neither lack of purification of the target DNA norshorter hybridization time inhibited the binding of probe to the target,the retention of hybridized probe by the filter, or the specificity ofthe reaction.

In another set of experiments designed to further test whether or nothybridization is responsible for retention of the probe by theultrafiltration membrane, either the probe or the target nucleic acid(but not both) was exposed to nuclease. In the first such experiment,the RNA probe was incubated with 5 μg of Ribonuclease A for one hour at37° C. prior to use as a probe. The results are presented in TABLE IV.In another such experiment, cell lysates were made in a buffer optimizedfor the activity of deoxyribonuclease I and treated the crude lysatewith 10 units of a commercial preparation of this enzyme (certified tobe NRase free) for one hour at 37° C. TABLE V shows the results of thisexperiment.

                  TABLE IV                                                        ______________________________________                                        RNase TREATMENT                                                               B. fragilis                                                                   Time of                                                                       reaction   Tube #1,     Tube #2,                                              in Min.    no RNase added                                                                             Probe RNase Treated                                   ______________________________________                                         5         0.68         0.14                                                  10         0.90         0.18                                                  15         1.00         0.21                                                  ______________________________________                                    

                  TABLE V                                                         ______________________________________                                        DNase TREATMENT                                                               B. fragilis                                                                   Time of                                                                       reaction         Tube #1,     Tube #2,                                        in Min.          no DNase added                                                                             DNase added                                     ______________________________________                                        5                0.15         0.08                                            15               0.16         0.07                                            30               0.19         0.08                                            60               0.23         0.08                                            90               0.27         0.08                                            19     hr.       0.75         0.11                                            ______________________________________                                    

Readings are ^(OD) 405, indicating extent of enzyme catalyzedcolorimetric reaction. Hybridization time: 30 minutes.

These data (as presented in TABLES IV and V) indicate that nucleic acidhybridization causes probe retention by the ultrafiltration membranesince the destruction of either component of the hybridization mixtureblocks filter retention of the probe.

EXAMPLE 3 HYBRIDIZATION OF PROBE cDNA WITH VARIOUS BACTERIAL NUCLEICACIDS AND ULTRAFILTRATION TO ISOLATE THE HYBRIDIZED MATERIAL

In this example, a copy DNA (cDNA) probe made by reverse transcriptionof bacterial ribosomal RNA (rRNA) was utilized. We purchased radioactivecDNA from Gen-Probe (Mycoplasma T. C. Detection Kit Catalog No. 1004from Gen-Probe, San Diego, California) and used this as probe in mostexperiments. Another probe was made using a procedure similar to thatdisclosed in Gen-Probe's patent application (Kohne, D. E., 1984, PatentCooperation Treaty WO 84/02721) but labelled with an enzyme rather thanwith radioactive label. Briefly, the Gen-Probe procedure uses purifiedrRNA as a template for the synthesis of cDNA using reversetranscriptase. The Gen-Probe kit contains cDNA made from MycoplasmarRNA. The enzyme labelled probe was fabricated using 1400 units ofcloned Moloney Murine Leukemia Virus reverse transcriptase (Catalog#8025SA) purchased from Bethesda Research Laboratories per 7 μg of B.subtilis rRNA. The specificity of both probes seems similar, they bindto target rRNA of all prokaryotic species tested.

These experiments allowed a comparison between the filter retentionmethod of separation with hydroxylapatite separation employed by theGen-Probe Mycoplasma T. C. detection kit. The single-stranded cDNA probehas homology with all prokaryotic ribosomal sequences tested, we used B.subtilis crude lysates as a source of rRNA target nucleic acid. Theprobe contains tritium-labelled nucleotides and detection uses ascintillaton counter. TABLE VI shows data from side-by-side comparisonof the two methods. Hybridization followed the detection kit labelinstructions and was carried out in purely aqueous solution at 72° C.The only difference between these two was in separation of hybridizedfrom unhybridized probe. The ultrafiltration membrane used in theseexperiments was an Amicon YM100, a cellulosic membrane having a MWCO of100,000. All data is given in counts per minute. Duplicate lysatesamples were made. Positive and negative controls were provided in thekit. It was noted that the positive control failed to develop a signalwith the filter separation method.

                                      TABLE VI                                    __________________________________________________________________________    Ultrafiltration on a Cellulosic Membrane With an MWCO of                      100,000 Versus Hydroxylapatite Method Using a Radioactive-Labelled            Probe With a Nucleic Acid Target Consisting of B. subtilis Crude Lysate       Separation Method                                                             Ultrafiltration       Hydroxylapatite                                         Sterile Positive                                                                           Negative Sterile                                                                           Positive                                                                           Negative                                       Media   Control                                                                            Control                                                                            Lysate                                                                            Media                                                                             Control                                                                            Control                                                                            Lysate                                    __________________________________________________________________________    CPM*                                                                              137 202  44   2644                                                                              230 2308 348   544                                                        2456              1089                                      __________________________________________________________________________     Total Counts Added: 6170                                                      Background: 20                                                                *Counts per minute of radioactivity.                                     

The data presented in TABLE VI demonstrate the utility ofultrafiltration membrane separation of hybridized from unhybridizedprobe using a very different probe, as well as its utility with a probelabelled with radioactivity.

In another set of experiments, various membrane filters were tested andcompared using this radioactive cDNA probe. In this experiment we usedan XM 300 membrane (used in the first and second example withenzyme-labelled probe) and a polysulfone membrane supplied by Schleicher& Schuell with an MWCO of 1,000,000. TABLE VII shows the results of thisexperiment. Data is shown in counts per minutes The target nucleic acidis B. subtilis crude lysate.

                  TABLE VII                                                       ______________________________________                                        Filter     Membrane                                                           Membrane Type                                                                            Pore Size     Lysate  CPM* Retained                                ______________________________________                                        acrylic    300,000  MWCO     none    233                                      acrylic    300,000  MWCO     present                                                                             6,325                                      polysulfone                                                                              1,000,000                                                                              MWCO     none    24                                       polysulfone                                                                              1,000,000                                                                              MWCO     present                                                                             4,184                                      ______________________________________                                         Total counts added: 13,316                                                    *Counts per minute.                                                      

These data, when combined with the data from TABLE VI, show thatmembranes of cellulosic, acrylic, or polysulfone construction and withMWCO ranges of from 100,000 to 1,000,000 can be used to separatehybridized from unhybridized nucleic acid.

In another set of experiments, a cDNA probe with an enzyme-labelledsignal system was tested with the ultrafiltration system of the presentinvention. In the same experiment a hybridization competition betweenthe probe cDNA probe and an unlabelled cDNA reduces the amount of proberetained by the filter The presence of nucleic acid of the same sequenceas the probe should reduce the signal and therefore the amount of proberetained by the filter if hybridization of probe to target rRNA iscausing probe retention by the filter. The unlabelled nucleic acidcompetes with the probe for hybridization sites on the target nucleicacid. In this experiment 10 μg of purified rRNA was used as the targetand the probe consisted of 0.42 μg of enzyme-labelled cDNA The 10 μg ofcompetitive cDNA was from the same preparation as the probe, but noenzyme had been attached to it The probe and competitive cDNA were addedsimultaneously to the target rRNA. Hybridizations were carried out inaccordance with the procedure described in EXAMPLE 2. The results arepresented in TABLE VIII. Results are in ^(OD) 405 after 20 minutes ofperoxidase reaction using a hybridization rime of 30 minutes.

                  TABLE VIII                                                      ______________________________________                                        Target        Competitive                                                     rRNA          cDNA       OD*.sub.405                                          ______________________________________                                        -             -          0.13                                                 +             -          0.99                                                 +             +          0.32                                                 ______________________________________                                         *Optical density at an incident light of 405.sub.nm wavelength using a        Spec 20 spectrophotometer with a 1 cm path length is a measurement of         color change due to enzyme activity.                                          + denotes additive is present.                                                - denotes additive is absent.                                            

This experiment demonstrates that the ultrafiltration method serves toseparate hybridized from unhybridized probe of a variety of types,labelled with both enzymes and radioactivity. It also adds more data tosupport the hypothesis that hybridization is indeed the most likelyexplanation for retention of probe by the filter since competition withan unlabelled probe significantly reduced the measured ^(OD) 405 form0.99 (without competitive cDNA) down to 0.32 ) with competitive cDNA).

What is claimed is:
 1. A method for identifying the presence of specificnucleic acid target molecules by virtue of specific base sequences onthe target molecules which comprises:(a) providing lysed single-strandedtarget molecules by lysing and denaturing cells of viruses containingDNA or RNA, (b) providing single-stranded labelled nucleic acid probemolecules having between 25 bases and about 10,000 per molecule andhaving an essentially complementary base sequence to a defined region insaid single-stranded target molecules, (c) hybridizing at least aportion of said single-stranded labelled nucleic acid probe molecules tosaid defined region in at least a portion of said target molecules,thereby forming a mixture of hybridized molecules and single-strandedmolecules, (d) ultrafiltering said mixture, using a membrane filterhaving a molecular weight cutoff of between about 50,000 and about1,000,000 daltons to cause said single-stranded nucleic acid moleculesto pass through said filter and said double-stranded molecules to beretained on said filter, and (e) measuring the amount of said portion ofsaid single-stranded labelled nucleic acid probe molecules in saidhybridized molecules or the amount of single-stranded labelled probemolecules that pass through said membrane filter and did not hybridizewith target nucleic acid molecules.
 2. The method of claim 1 whereinsaid molecular weight cutoff is between about 100,000 and about 500,000daltons and wherein said probe molecules have no more than about 5,000bases per molecule.
 3. The method of claim 2 wherein said molecularweight cutoff is between about 200,000 and about 300,000 daltons.